Biological molecules (biomolecules) have three-dimensional structure or conformation, and rely on this structure for their biological activity and properties. Examples of such biomolecules include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and proteins. These biomolecules are essential for life, and represent therapeutic agents and targets in treating various medical diseases and conditions. Proteins represent a broad class of biomolecules. Different classes of proteins such as enzymes, growth factors, receptors, antibodies, and signaling molecules depend on their conformational structure for their biological activity. Other classes of proteins are primarily structural, e.g. collagen and cartilage, and do not possess biological activity per se.
Exposing biomolecules to various environments such as variations in pH, temperature, solvents, osmolality, etc., can irreversibly change or denature the conformational state of the biomolecule, rendering it biologically inactive. Some of the mechanisms involved in the deactivation of these biomolecules include aggregation, oxidation, various types of bond cleavage including hydrolysis and deamidation, and various types of bond formation, including cross-linking and other covalent binding, for example the rearrangement of disulfide bonds.
Bone morphogenetic proteins and the closely related growth and differentiation factors (in both monomeric and dimeric forms) belong to the TGF-β superfamily of proteins. This class of proteins includes members of the family of bone morphogenetic proteins that were initially identified by their ability to induce ectopic endochondral bone formation (see Cheng et al. “Osteogenic activity of the fourteen types of human bone morphogenic proteins” J. Bone Joint Surg. Am. 85A: 1544-52 (2003)). There are alternate names for several of these proteins, (see Lories et al., Cytokine Growth Factor Rev 16:287-98 (2005)). All members of this family share common structural features, including a carboxy terminal active domain, and are approximately 97-106 amino acids in mature length. All members share a highly conserved pattern of cysteine residues that create 3 intramolecular disulfide bonds and one intermolecular disulfide bond. The active form can be either a disulfide-bonded homodimer of a single family member or a heterodimer of two different members. (see Massague Annu. Rev. Cell Biol. 6:957 (1990); Sampath, et al. J. Biol. Chem. 265:13198 (1990); Ozkaynak et al. EMBO J. 9:2085-93 (1990); Wharton, et al. PNAS 88:9214-18 (1991); Celeste et al. PNAS 87:9843-47 (1990); Lyons et al. PNAS 86:4554-58 (1989), U.S. Pat. No. 5,011,691, and U.S. Pat. No. 5,266,683).
It is well established that many sugars stabilize biomolecules in solution and afford protection to isolated cells and biomolecules. These compounds are well established as cryoprotectants and osmoregulators in various species (see Yancey J. Exper. Biol. 208: 2819-30 (2005)). In the development of lyophilized pharmaceutical proteins, sugars (saccharides and polyols) are often added to the formulation in order to improve the stability of the protein and prolong the shelf life. There are two main theories on the mechanism of the stabilizing action of sugars: 1) the sugar excipients serve to dilute the proteins in the solid state, thereby decreasing protein-protein interactions and preventing molecular degradation, such as aggregation, and 2) the sugar excipients provide a glassy matrix wherein protein mobility and hence reactivity are minimized. In both of these mechanisms, it is critical that the sugar remains in the amorphous, protein-contacting phase. Various environmental factors, such as increased temperature and moisture, can induce sugar crystallization. Thus, it is important to optimize the conditions and materials used to suit the particular biomolecule and system under consideration.
Lyophilization (freeze-drying) is a method commonly used to preserve biomolecules. Freeze-drying is generally thought to be more disruptive to the biological activity of biomolecules than freeze-thawing or temperature-induced denaturation. The magnitude of damage varies considerably with different biomolecules and different conditions, and various investigators have studied different systems. The freezing of aqueous solutions creates an initial increase in solute concentrations that can be more damaging to labile compounds than the freezing itself. Excipients such as sugars, proteins, polymers, buffers, and surfactants can be added to stabilize the activity of the biomolecule, but have limited and varying degrees of success, depending on the system. Crowe, et al. describes the stabilization of dry phospholipid bilayers and proteins by sugars (Biochem. J. 242: 1-10 (1987)), and also reviews the recent understanding of the mechanisms of trehalose stabilization of cells in “The trehalose myth revisited: Introduction to a symposium on stabilization of cells in the dry state” Cryobiology 43, 89-105 (2001). The current thinking is that there are two separate and different requirements for maintaining a viable and useful lyophilized protein: 1) the protein must be protected during the freezing process, and 2) the protein must be protected during the subsequent drying and reconstitution. These are different requirements that are not necessarily met by any one excipient or set of conditions.
Various researchers have reported on using various excipients to protect various biomolecules, for example Gloger, et al. (Intl. J. Pharm. 260: 59-68 (2003)) described the lyoprotection of aviscumine using low molecular weight dextrans to stabilize the protein, and showed that the buffer system and polysorbate 80 alone are suitable to protect the protein during freezing, but dextran is needed to protect the protein during drying; Goodnough, et al. (Appl. Env. Biol. 58(10: 3426-28 (1992)) investigated the stabilization of Botulinum toxin type A during lyophilization using serum albumin as stabilizer and various other excipients, and reported that none of the excipients had any beneficial effect, but by eliminating NaCl from the lyophilization mixture and by controlling the pH, the recovery of active toxin was dramatically improved; Costantino, et al. (J. Pharm. Sci. 87(11): 1412-20 (1998)) described the effects of various saccharides on the stability and structure of lyophilized recombinant human growth hormone, and showed that all of the excipients tested significantly improved the stability of the protein; Ramos et al. (Appl. Envir. Microbiol. 63(10): 4020-25 (1997)) showed that 2-O-β-mannosylglycerate is effective in protecting several dehydrogenase enzymes isolated from various sources from thermal stress, and that the protection afforded by 2-O-β-mannosylglycerate was similar to or superior to trehalose for all of the enzymes studied, but was not effective in protecting glutamate dehydrogenase isolated from P. furiosis; Brus, et al. (J. Control. Rel. 95:119-31 (2004)) investigated the stabilization of oligonucleotide-polyethylenimine (PEI) complexes by freeze-drying, and reported that these complexes did not benefit from the addition of sugars such as sucrose or trehalose, but that plasmid-PEI complexes did benefit from the addition of such sugars. These investigators report varying degrees of success, as measured by various methods on various biomolecules. None of these investigators have reported on the protection of BMP's.
Thus, there is conflicting evidence on what is an optimal combination of excipients to afford lyoprotection of biomolecules. There is not any one combination of excipients that is optimal for all biomolecules, but rather a significant degree of experimentation is required to obtain the desired results for the biomolecule under investigation. There remains a need for a pharmaceutically acceptable combination of excipients to protect BMP's during lyophilization, storage, and use.